Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

Current and Potential Biofuel Production from Plant Oils

Environmental concerns and depletion of fossil fuels along with government policies have led to the search for alternative fuels from various renewable and sustainable feedstocks. This review provides a critical overview of the chemical composition of common commercial plant oils, i.e., palm oil, olive oil, rapeseed oil, castor oil, WCO, and CTO and their recent trends toward potential biofuel production. Plant oils with a high energy content are primarily composed of triglycerides (generally >?95%), accompanied by diglycerides, monoglycerides, and free fatty acids. The heat content of plant oils is close to 90% for diesel fuels. The oxygen content is the most important difference in chemical composition between fossil oils and plant oils. Triglycerides can even be used directly in diesel engines. However, their high viscosity, low volatility, and poor cold flow properties can lead to engine problems. These problems require that plant oils need to be upgraded if they are to be used as a fuel in conventional diesel engines. Biodiesel, biooil, and renewable diesel are the three major biofuels obtained from plant oils. The main constraint associated with the production of biodiesel is the cost and sustainability of the feedstock. The renewable diesel obtained from crude tall oil is more sustainable than biofuels obtained from other feedstocks. The fuel properties of renewable diesel are similar to those of fossil fuels with reduced greenhouse gas emissions. In this review, the chemical composition of common commercial plant oils, i.e., palm oil, olive oil, rapeseed oil, castor oil, and tall oil, are presented. Both their major and minor components are discussed. Their compositions and fuel properties are compared to both fossil fuels and biofuels.     

七种植物籽油中脂肪酸成分的比较分析(英文)

为寻求新的食用油资源,发展了一种快速可靠的气相色谱-质谱联用方法,用于植物籽油中脂肪酸成分的定性鉴定和含量测定。所建立的方法成功用于葡萄籽、南瓜籽和猕猴桃籽等七种植物籽油中的棕榈酸、十八烷酸、油酸、亚油酸和α-亚麻酸的定性定量分析。结果表明,刺葡萄籽油、普通葡萄籽油、国外葡萄籽油、南瓜籽油、枸杞籽油和西番莲籽油均具有相似的脂肪酸谱,尽管其中它们所含上述五种脂肪酸含量不同,由于均存在人体所必需的饱和与不饱和脂肪酸,故可以用作替代食用油。猕猴桃籽油因为其存在高含量的α-亚麻酸成分,可能是更好的食用油和营养油资源。本文首次对枸杞籽油、西番莲籽油和猕猴桃籽油脂肪酸成分进行绝对含量分析,为新的食用油资源的开发提供了重要的依据。     

A Rapid and Simple Method for Staining Lipid in Fixed Nematodes

A method is described for staining lipid in fourth-stage dispersal juvenile nematodes fixed with formal-acetic fixative (FA4:1). Bursaphelenchus xylophilus fourth-stage dispersal juveniles were fixed with hot FA4:1 for 24 hours, excess fixative was removed, and a solution of saturated oil red O in 96% ethanol added and allowed to sit for 25 minutes at 60 C. Excess oil red O was removed, nematodes were washed twice with 70% ethanol, and were processed to pure glycerin. Lipid droplets within the nematodes were viewed by light microscopy and appeared as dark red spheres of various sizes. Computerized image analysis was used to quantify lipid droplet area.     

11种植物精油对6种植物病原真菌的抑菌活性研究

为筛选有效的植物杀菌成分,采用菌丝生长法,测定了香茅油、薰衣草油、菊花油、月桂油、柠檬油、广藿香油、肉桂油、天竺葵油、迷迭香油、茶树油、薄荷油对6种植物病原真菌的抑菌活性。发现在2 g/L的浓度下,上述11种精油对6种供试病菌均有明显的抑制作用,其中香茅油、肉桂油、天竺葵油、月桂油、茶树油和薄荷油对6种病原真菌的抑制率均为100%。剂量效应试验表明,肉桂油对灰葡萄孢（Botrytis cinerea）和禾谷镰孢菌（Fusariumgraminearum）的EC50值分别为29.05μg/mL和42.96μg/mL,而天竺葵油对灰葡萄孢（Botrytis cinerea）和禾谷镰孢菌（Fusarium graminearum）的EC50值分别为34.02μg/mL和68.48μg/mL。     

The Recognition of Lipolytic Activity with the Microscope

In view of its probable wide applicability, it seems desirable to publish a note on a simple technic for the recognition with the microscope of the action of lipase. In brief, the method is to make an emulsion of neutral fat previously stained with a red Sudan stain, subject some of the emulsion to the action of the supposed lipase for an appropriate time and then examine with the microscope a recovered drop of the emulsion in a solution of Nile blue sulfate. It has long been known that Nile blue sulfate stains liquid neutral fats a reddish color and fatty acids blue.     

Staining Sections Coated with Radiographic Emulsion; A Nuclear Fast Red, Indigo-Carmine Sequence

The radioautographs consisting of emulsion-coated sections, after development, fixation and washing, are stained 3 min (uncritical) by an aqueous solution of 5% aluminium sulfate containing 0.1% nuclear fast red, then washed 2-5 min, and stained 5-10 sec by a saturated aqueous solution of picric acid to which 0.25% of indigo-carmine had been added. This technic stains differentially cell nuclei, cytoplasm, muscle fibers, connective tissues and borders of some epithelial cells. It is unnecessary to subject the slides to differentiating solutions.     

Nematicidal Activity of Plant Essential Oils against Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

The nematicidal activity and poisoning symptoms of 88 plant essential oils against Bursaphelenchus xylophilus were examined by an immersion bioassay. Results were compared with those of three trunk-injection nematicides: fenitrithion, levamisol hydrochloride, and morantel tartrate. As judged by 24 h LC₅₀ values, cinnamon bark oil (0.12 mg/ml) was the most effective nematicide, followed by coriander herb oil (0.14 mg/ml). Potent nematicidal activity was also observed with lemongrass, oregano, thyme red, and clove bud oils (LC₅₀, 0.57-0.88 mg/ml). Fenitrothion was ineffective (LC₅₀, > 10 mg/ml). In typical poisoning symptoms in B. xylophilus, these essential oils exerted rapid nematicidal action and the nematodes killed usually showed an extended shape, whereas levamisole hydrochloride and morantel tartrate usually exhibited semicircular and coiling shapes, respectively. The essential oils described merit further study as botanical nematicides for the control of pine wilt disease caused by B. xylophilus.     

Rapid Single-Solution Polychrome Staining of Semithin Epoxy Sections using Polyethylene Glycol 200 (PEG 200) as a Stain Solvent

Rapid, onestep polychromatic staining of 0.75-1.5 μm epoxy sections of glutaraldehyde-osmium fixed tissues can be obtained with mixtures of basic fucbsin and toluidme blue O in alkaline polyethylene glycol ZOO (PEG ZOO). Sections are attached to slides by heating at 100 C for 45 seconds and stained at that temperature for 2-3 minutes with a solution consisting of PEG 200 (50 ml), 0.2 N KOH (0.75 ml), basic fuchsin (1.7 gm), and toluidine blue O (0.3 gm). Red-blue balance and selective staining of different structures can be controlled by varying the amount of toluidine blue added. After rinsing with 10% acetone and rapid drying, sections are covered with immersion oil or mounting medium and a cover-slip. Total time from cutting of a section to finished preparation is less than 6 minutes. This staining solution is stable, does not produce precipitates on the sections, and does not wrinkle or lift the sections from the slides.     

Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.     

Sudan Black B And Acetocarmine as a Combination Stain

Epidermis stripped from either fresh or fixed plant organs, or sections of paraffin-embedded or fresh material are placed on a slide and covered with a drop or two of iron-acetocarmine. The stain is intensified by warming the slide over a flame. After a few minutes a drop or two of a saturated solution of Sudan black B in 45% acetic acid is added and a cover slip applied. The preparations cannot be made permanent, but last a few weeks if sealed with a compound such as gum mastic-paraffin, or if the combined stain is drained off and a drop of Karo syrup is added before the cover slip is applied. The acetocarmine produces its usual staining effects, i.e., nuclei dark red and some components of the cytoplasm of certain cells a less intense red. The Sudan black B colors lipid structures an intense blue.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

A Differential Staining Method for Elastic Fibers, Collagenic Fibers, and Keratin

A method allowing for the differential presentation of elastic fibers, other connective tissue fibers, epithelial and other types of cytoplasm, and keratin is described. The procedure is based on the affinity of orcein for elastic fibers, of anilin blue for collagenic material, and of orange G for keratin. Bouin-fixed, tissue-mat embedded sections are stained in Pinkus' acid orcein for 1 1/2 hours and rinsed in distilled water. The sections are differentiated in 50% alcohol containing 1% hydrochloric acid, washed in tap and then in distilled water. The sections are next transferred for I to 2 minutes to the anilin blue, orange G, phosphomolybdic acid combination known as solution No. 2 of Mallory's connective tissue stain, diluted 1:1 with distilled water. They are then rinsed in distilled water, quickly passed into 95% alcohol, and dehydrated in absolute alcohol containing some orange G, after which they are cleared and mounted. Within less than two hours sections may be stained and mounted with the following results: elastic fibers — red; collagenic fibers — blue; muscle fibers — yellow; keratin — orange.     

New Uses for Calcium Chloride Solution as a Mounting Medium

Fresh cross sections of stems [Psilotum nudum, Coleus blumei, and Pelargonium peltatum] and roots (Setcreasea purpurea) 120 μm thick were fixed in FPA₅₀ (formalin: propionic acid: 50% ethanol, 5:5:90, v/v) for 24 hr and stored in 70% ethanol. The sections were transferred to water and then to 1% phloroglucin in 20% calcium chloride solution plus either hydrochloric, nitric, or lactic acid in the following ratios of phloroglucin-CaCl₂ solution:acid: 25:4, 20:2, or 15:5. The sections were mounted on slides either in one of the three mixtures or in fresh 20% calcium chloride solution. A rapid reaction of the acid-phloroglucin with lignin produced a deep red color in tracheary elements and an orange-red color in sclerenchyma. Fixed and stored leaf pieces from Nymphaea odorata were autoclaved in lactic acid, washed in two changes of 95% ethanol, transferred to water, and treated with the three acid-phloroglucin-calcium chloride mixtures. The abundant astrosclereids stained an orange-red color similar to that of sclerenchyma in the sections. In addition, a new method is reported for specifically staining lignified tissues. When sections or leaf pieces are stained in aqueous 0.05% toluidine blue O, then placed in 20% calcium chloride solution, all tissues destain except those with lignified or partially lignified cell walls. Thus, toluidine blue O applied as described becomes a reliable specific test for lignin comparable to the acid-phloroglucin test.     

A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Thin (0.5-1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2-5 min. The water solution was more effective for some epoxy sections (10-80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5-10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1-30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.     

Staining Residual Lipids in Ultrathin Sections of Tissues Embedded in Polyester Resin

Rat suprarenal glands fixed in Palade's 1% OsO₄, buffered at pH 7.7 with veronal-acetate, to which 0.1% MgCl₂ was added, were embedded in Vestopal-W and sectioned at 0.2-1 µ. The sections were attached to slides by floating on water, without adhesive, and drying at 60-80° C, placed in acetone for 1 min and then treated with the following staining procedure: Place the preparation in a filtered solution of oil red O, 1 gm; 70% alcohol, 50 ml; and acetone, C.P., 50 ml; for 0.5-1 hr. Rinse in absolute ethyl alcohol; drain; counterstain with 0.5% aqueous thionin for 5 min; rinse in distilled water; drain; stain in 0.2% azure B in phosphate buffer at pH 9, for 5 min. Dry and apply a drop of immersion oil directly on the section. The preparations are temporary. Ciaccio-positive lipids, rendered insoluble by OsO, fixation, stained red to ochre.     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造, 进而生产有益于健康的食用油所取得的进展。近10年来, 应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展, 随着植物脂肪酸生物合成途径的日趋明确, 通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油。     

基因工程在改善植物油营养价值中的应用

本文介绍了近年来应用基因工程技术对植物油的脂肪酸成分进行改造,进而生产有益于健康的食用油所取得的进展.近10年来,应用基因修饰的方法对植物油进行营养学方面的改良已经取得了很大的进展,随着植物脂肪酸生物合成途径的日趋明确,通过转基因技术可以让植物生产许多含有特殊脂肪酸成分且对人类健康有益的植物油.     

植物精油对烟草甲害虫的毒力测定

采用密闭熏蒸方法,测定了多种植物精油对烟草甲害虫的熏杀活性.结果表明供试植物精油之间对同一害虫存在着较大差异,而供试精油桉叶油、大叶留兰香油、小叶留兰香油、冬青油对该种害虫具有很高的熏杀毒力.植物精油是极有开发利用前景的植物性杀虫剂.